The current understanding of alphavirus and flavivirus life cycles at the molecular level is limited because we lack a structural foundation for the viral and host proteins, especially those involved in RNA replication, genome packaging and particle assembly. Viruses within these two groups pose significant risks to large segments ofthe population and methods for controlling infection and disease are few. Therefore, we propose to continue our systematic collaborative investigation that will provide a comparative approach to the structure and function of the virally encoded proteins representing the spectrum of pathogens within these two groups. In this proposal, we will shift our emphasis to encompass not only viral proteins but also host proteins and the appropriate complexes beh/veen host and pathogen. The tools proposed for studying the structure and function of these viral proteins are varied and multi-disciplinary. We previously placed a heavy emphasis on X-ray crystallography of individual viral proteins and intact viruses (Michael Rossmann, Janet Smith). In addition to our emphasis on high-resolution structures from crystallography, a key role is played by Tim Baker, Wen Jiang, and Michael Rossmann in the cryo-electron microscopy study of viruses, complexes of receptors and antibodies, and intermediates In their assembly/disassembly. Electron tomography and immuno-electron microscopy will also be employed as a means to image sub-cellular structures induced and/or utilized during virus infection. Functional biochemical and genetic analyses (Richard Kuhn, Janet Smith) will complement and support the structural studies. Site-directed mutagenesis and protein interaction approaches to explore interactions found, or hypothesized, on the basis of structure determination will be emphasized to probe various aspects of the virus life cycle. These approaches will be integrated and an iterative process will be designed for extracting optimal information and refining experimental directions. The synergy of individual projects, which employ crystallography, cryo-electron microscopy, biochemistry, molecular biology, and genetics, in a highly collaborative environment is a critical strength of our proposal. This structural-functional approach should provide a wealth of information for understanding the life cycle and pathogenesis of these viruses that will be invaluable in the design of vaccines and antivirals, as well as providing a knowledge base should there be an emergency related to the emergence or reemergence of these viruses.